B. burgdorferi, the agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), nitrogen dioxide (NO2), nitrogen trioxide (N2O3) and peroxynitrite (NO3). There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs as the bacteria migrate through the tick salivary during transmission. Our lab has demonstrated that the salivary glands of Ixodes scapularis contain significant levels of ROS and RNS during feeding. Additionally, we have observed ROS and RNS in the tick midgut during feeding. Not only does ROS and RNS present a significant challenge to B. burgdorferi during transmission but reactive oxygen and nitrogen intermediates could act as signals to modulate gene expression through BosR. To understand these processes, we have taken approaches that (1) have identified the intracellular targets of ROS and RNS in B. burgdorferi, (2) have identified some of the enzymatic function of key proteins that protect the bacterium from ROS and RNS, and (3) have identified key proteins (e.g., Borrelia oxidative stress regulator, BosR, RpoN, RpoS, and Rrp2) which data suggests are involved in regulating key defense enzymes and important virulence factors e.g., outer surface protein C (OspC), etc. First, unlike most bacteria, the major intracellular targets of ROS are the membrane lipids and lipoproteins while those of RNS are zinc metalloproteins. Second, key ROS defense enzymes include a Mn-dependent superoxide dismutase (SOD), a Dps/Dpr homologue (NapA/BicA) which functions as a thioredoxin-dependent peroxiredoxin, CoA disulfide reductase (CoADR), thioredoxin (Trx) and thioredoxin reductase (TrxR). The cellular defenses against RNS have begun to be elucidated and recent data suggests the Base Excision Repair (BER), Nucleotide Excision Repair (NER) and Mismatch Excision Repair (MMS) systems play a key roles in protecting B. burgdorferi DNA from RNS and ROS only in the tick midgut during feeding. The systems are not requires for survival in mice. Finally, it has been shown that BosR, regulates the genes encoding SodA (Mn-SOD), NapA and CoADR) in response to ROS. As important, recent analyses of BosR mutants in our and other laboratories have demonstrated that BosR has a positive regulatory effect on RpoS (and OspC) through the RpoN-RpoS regulatory cascade. Ouyang et al have identified a BosR binding sequence (BosR box), adjacent to the RpoN promoter upstream of rpoS. Clearly, the BosR-dependent regulation of the RpoN-RpoS regulatory cascade is key to the pathogenesis of B. burgdorferi. B. burgdorferi's ability to adapt and survive in very different environments (tick versus mammal)is attributed to its ability to sense changes in temperature, pH, cell density, oxygen, manganese and/or exposure to host factors and alter gene expression accordingly. Previous reports have demonstrated that central to the regulation of these responses are the sigma factors, RpoN and RpoS. Importantly, RpoN-dependent regulation of RpoS is responsible for the expression of key virulence factors e.g., outer surface protein C (OspC), OspA and decorin-binding protein A (DbpA0 required for infectivity and transmission during the infective cycle. The activities of RpoN are tightly controlled and require ATP-dependent activation. Many of the activators of RpoN-RNA polymerase (RNAP) are response regulators (RR) of two-component regulatory systems and phosphorylation of these RR results in their activation. These RRs are phosphorylated by small molecular weight phosphate donors (e.g., ATP; designated as auto-phosphorylation) or, more commonly, by their cognate protein histidine kinase (HK) in response to an environmental signal. The activator of RpoN in B. burgdorferi, Rrp2 encoded by bb0763 (rrp2), is also a RR of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, Hk2 encoded by bb0764 (hk2). Most RpoN transcription activators have a well-defined protein structure consisting of three functional domains: (1) the N-terminal, receiver domain which is phosphorylated by its histidine kinase; (2) a central domain that binds to RpoN-RNAP holoenzyme and hydrolyzes ATP to generate an RpoN-RNAP open complex that initiates transcription and; (3) the c-terminal DNA binding domain which recognizes enhancer-like elements to target RpoN-dependent promoters. While the receiver and central domains of Rrp2 function similar to other RpoN transcriptional activators, the DNA enhancer binding domain does not. Our group, has used numerous approaches to attempt to identify a potential Rrp2 enhancer sequence upstream of rpoS. Data from our and several groups suggest that Rrp2 activates transcription of the RpoN-dependent RpoS promoter in an enhancer independent fashion (1). Importantly, numerous attempts to inactivate rrp2 have failed leading to the speculation that Rrp2 was essential for cell viability. Very recently, Groshong et al. generated a conditional rrp2 mutant to show that Rrp2 was essential for cell growth and overexpression was lethal (2). More recently, Dr. Yangs and our research group have developed an assay to measure Rrp2 phosphorylation in vivo. While essential function of Rrp2 in cell physiology remains to be determined, it is clear that it plays a central role in transmission and pathogenesis of B. burgdorferi. To determine the influence of Hk2 on RpoS expression, an Hk2 mutant in B. burgdorferi low-passage strain B31-A3 was generated by deleting hk2. We postulated that Hk2 might be required for temperature-, pH- or cell-density dependent expression of rpoS mediated by Rrp2. Surprisingly, transcription fusions in wild-type and A3;delta hk2 backgrounds indicated that Hk2 was not responsible for temperature, pH, or cell density-dependent regulation of the RpoN-RpoS regulatory cascade by Rrp2. Since it had been shown that Rrp2 was required for these types of regulation in B. burgdorferi, it seemed likely that Rrp2 could be activated by another HK or a small molecular weight phosphate donor in order to mediate Rrp2-dependent regulation of the RpoN-RpoS regulatory cascade. Originally, we believed that the low molecular weight phosphate donor, acetyl phosphate (acetyl-P), played this role in this regulatory process. But more complete analyses of the acetate/mevelonate pathway that generates acetyl-P indicates that this may not be the case. Additionally, we do not know the exact role Hk2 plays in Rrp2-dependent regulation but recent data suggests that the putative phosphatase activity of Hk2 may be as, or more important than its kinase activity. Our current model suggests that Hk2 modulates the ratio of phosphorylated to non-phosphorylated Rrp2 (Rrp2-P:Rrp2). Since the active form of Rrp2 (a dimer of Rrp2-P) is required to stimulate RpoN-dependent transcription, Hk2 would, through its phosphatase activity, negatively affect Rrp2-dependen regulation in B. burgdorferi.